Liposomes are completely closed structures comprising lipid bilayer membranes containing an encapsulated aqueous volume. Liposomes may contain many concentric lipid bilayers separated by an aqueous phase (multilamellar vesicles or MLVs), or alternatively, they may comprise a single membrane bilayer (unilamellar vesicles). The lipid bilayer is composed of two lipid monolayers having a hydrophobic "tail" region and a hydrophilic "head" region. In the membrane bilayer, the hydrophobic (nonpolar) "tails" of the lipid monolayers orient toward the center of the bilayer, whereas the hydrophilic (polar) "heads" orient toward the aqueous phase. The basic structure of liposomes may be made by a variety of techniques known in the art.
Liposomes have typically been prepared using the process of Bangham et al., (1965 J. Mol. Biol., 13: 238-252), whereby lipids suspended in organic solvent are evaporated under reduced pressure to a dry film in a reaction vessel. An appropriate amount of aqueous phase is then added to the vessel and the mixture agitated. The mixture is then allowed to stand, essentially undisturbed for a time sufficient for the multilamellar vesicles to form. The aqueous phase entrapped within the liposomes may contain bioactive agents, for example drugs, hormones, proteins, dyes, vitamins, or imaging agents, among others.
Liposomes may be reproducibly prepared using a number of currently available techniques. The types of liposomes which may be produced using a number of these techniques include small unilamellar vesicles (SUVs) [See Papahadjapoulous and Miller, Biochem. Biophys. Acta., 135, p. 624-638 (1967)], reverse-phase evaporation vesicles (REV) [See U.S. Pat. No. 4,235,871 issued Nov. 25, 1980], stable plurilamellar vesicles (SPLV) [See U.S. Pat. No. 4,522,803, issued Jun. 11, 1985], and large unilamellar vesicles produced by an extrusion technique as described in copending U.S. patent application Ser. No. 622,690, filed Jun. 20, 1984, Cullis et.al., entitled "Extrusion Technique for Producing Unilamellar Vesicles", relevant portions of which are incorporated herein by reference.
Liposomes may be used as carriers for a wide variety of materials, for example drugs, cosmetics, diagnostic reagents and bioactive compounds, among others. Liposome compositions to which proteins are conjugated may be designed for both diagnostic and in vivo uses. For example, the ability to produce an antibody-directed vesicle would be a distinct advantage over similar undirected systems (Gregoriadis, G., Trends Pharmacol Sci, 4, p. 304-307, 1983), as would the targeting of a specific receptor or other cell surface feature. Useful applications of these protein-liposome conjugates would be in the selective targeting of cytotoxic compounds entrapped in vesicles to circulating tumor cells (Wolff et.al., Biochim. Biophys. Acta, 802, p. 259-273 1984), or applications of these immunoglobulin-associated vesicles in the development of diagnostic assays. Further applications could result from the targeting of a specific protein-receptor interaction for delivery of active agent to a specific site in a patient. Indeed, protein conjugated liposomes theoretically could be used to target the delivery of any active agent to a site in the patient's system to which the protein will bind. Numerous techniques for the conjugation of proteins to liposomes have already been developed for a variety of purposes including the targeting of drugs via immunoliposomes [See Leserman, et al., Nature, 288, 602 (1980), Heath, et al., Proc. Natl. Acad. Sci. USA, 80, 1377 (1983) and Huang, et al., J. Biol. Chem., 258, 14034 (1983)], diagnostic protocols [See Ishimori, et al., J. Immunol. Methods, 75, 351 (1984) and Rodney, et al., J. Immunol., 134, 4035 (1985)] and liposomal vaccines [See Allison, et al., Nature, 252, 252 (1974)].
Liposomes may be covalently coupled to proteins, antibodies and immunoglobulins. Heath et.al. (Biochim. Biophys. Acta., 640, p. 66-81, 1981), describe the covalent attachment of immunoglobulins to liposomes containing glycosphingolipid. Leserman et. al. (Liposome Technology, III, 1984, CRC Press, Inc., California, p. 29-40; Nature, 288, p. 602-604, 1980) and Martin et. al., (J. Biol. Chem., 257, p. 286-288, 1982) have described procedures whereby thiolated IgG or protein A is covalently attached to lipid vesicles, and thiolated antibodies and Fab' fragments are attached to liposomes, respectively. These protocols and various modifications (Martin et.al, Biochemistry, 20, p. 4229-4238, 1981; and Goundalkar et.al., J. Pharm. Pharmacol., 36, p. 465-466, 1984) represent the most versatile approaches to coupling. Avidin-coupled and avidin and biotinyl-coupled phospholid liposomes containing actinomycin D have successfully targeted tumor cells expressing ganglio-N-triosylceramide (Urdal et.al., J. Biol. Chem., 255, p. 10509-10516, 1980). Huang et.al. (Biochim. Biophys. Acta., 716, p. 140-150, 1982) demonstrate the binding of mouse monoclonal antibody to the major histocompatibility antigen H-2 (K), or goat antibody to the major glycoprotein of Molony Leukemia Virus, to palmitic acid. These fatty acid modified IgGs were incorporated into liposomes, and the binding of these liposomes to cells expressing the proper antigens characterized. Other in vitro efforts to promote specific binding of liposomes coated with specific immunoglobins have been performed (Sharkey et.al., Fed. Proc., 38, 1089, 1979). In still other coupling studies, Rahman et. al. found that tissue uptake of liposomes could be altered by attachment of glycolipids to the liposomes (J. Cell Biol., 83, p. 268a, 1979).
In accordance with a primary use for liposomes, the entrapment of antineoplastic agents inside liposomal bilayers has resulted in more efficacious therapy as compared to direct administration of the drug. (Forssen et.al., Cancer Res., 43, p. 546, 1983; and Gabizon et.al., Cancer Res., 42, p. 4734, 1982). A major problem with the encapsulation of antineoplastic drugs as well as other agents is that many of these drugs have been found to be rapidly released from liposomes after encapsulation. This is an undesirable effect, in view of the fact that toxicity of many of the antineoplastic agents can be significantly reduced through liposome encapsulation as compared to direct administration. See, for example, Forssen et.al. Cancer Res. 43, 546 (1983) and Rahman et.al. Cancer Res., 42, 1817 (1982). In addition, certain pharmacological agents which are favorably delivered in sustained release fashion are not accommodated by standard liposomal delivery systems; many liposomal compositions release the agent too rapidly to provide sustained release delivery.
One answer to the above-described problem is the use of preformed, stable liposomes which maintain the stability and sustained release characteristics of the liposomal system. Liposomal compositions comprising protein-coupled liposomes have produced storage stable liposomes which may be stored stably for an indefinite period, as described in U.S. patent application, Ser. No. 811,037, filed Dec. 18, 1985, entitled "Novel Composition for Targeting, Storing and Loading of Liposomes". These liposomes, which include streptavidin and immunoglobulin coupled to liposomes, may be stored in a dehydrated state, with loading of the liposomes on an "as needed" basis. These protein-coupled liposomes have been loaded with ionizable antineoplastic agents wherein a transmembrane potential is created across the walls of the liposomes and the antineoplastic agent is loaded into the liposomes by means of the transmembrane potential. See, for example, U.S. patent application Ser. No. 749,161, Bally et.al. entitled "Encapsulation of Antineoplastic Agents in Liposomes," filed Jun. 26, 1985 and U.S. patent application Ser. No. 941,913, entitled "Novel Composition for the Targeting, Storing and Loading of Liposomes," filed Dec. 15, 1986, relevant portions of which are incorporated herein by reference.
As explained above, protein-liposome conjugates have many potential applications, ranging from diagnostic systems to the targeting of disease states in vivo. As indicated elsewhere [Loughery, et al., Biochim. Biophys. Acta., 901, 157 (1987], the coupling of streptavidin to liposomes results in a flexible basic system which subsequently allows the straightforward conjugation of a variety of proteins.
Covalent attachment of liposomes to antibodies which are directed against cell surface antigens such as those associated with transformed cells, has considerable therapeutic potential. However, at the present time, such targeted liposomal systems have mainly been used for in vitro applications such as in diagnostic assays (Martin and Kung, Ann. N.Y. Acad. Sci., pp. 443-449 (1985). In order to exploit the full potential of antibody targeted carrier systems, as well as other systems, for example liposome-protein coupling and liposome-cofactor coupling, an improved versatile and reliable methodology for coupling should be developed.
To date, no general procedure for attaching proteins, antibodies and other molecules to liposomes is yet available. Leserman, et al. Nature (London), 288, 602-604 (1980) and Barbet, et al., J. Supramol. Struct. Cell. Biochem., 16, 243-258 (1981) have described a procedure wherein a thiolated IgG is covalently attached to liposomes containing N-[3-(2-pyridyldithio)-propionyl]phosphatidylethanolamine (PDP-PE) via a disulfide bond. A more general version of this procedure was developed by coupling protein A to vesicles (see, for example, Leserman (1980), supra.), which takes advantage of the ability of protein A to bind the Fc protion of IgGs of certain classes. One major limitation of this method is that many monoclonal antibodies are not of the appropriate class.
An alternative to the above approach to coupling is that of Martin and Papahajopoulos, J. Biol. Chem., 257, 286-288 (1982) who developed the technique of covalently attaching antibodies and Fab' fragments to liposomes containing N-[4-(p-maleimidophenyl)-butyryl]phosphatidylethanolamine (MPB-PE) by formation of a thio-ether linkage with the maleimido group, a linkage which is considerably less susceptible to reducing conditions found in the serum than is the disulfide linkage of the Leserman method. The Martin/Papahajopoulos approach as well as various modifications of this approach [see, for example, Wolff and Gregoriadis, Biochem. Biophys Acta, 802, 259 (1984), Martin, et al., Biochemistry, 20, 4229 (1981) and Goundalkar, et al., J. Pharm. Pharmacol., 36, 465 (1984) represent the most versatile approaches to coupling currently available.
In this Inethod of cross-linking liposomes to proteins, antibodies, cofactors and other molecules to liposomes, cross-linking agents containing a maleimide group, for example, N-succinimidyl 4-(p-maleimidophenyl)butyrate (SMPB), among others, are used to cross-link phosphatidylethanolamine and other amine containing lipids to thiol containing conjugated molecules, for example, proteins, antibodies, cofactors and other molecules containing reactive thiols. Prior art cross-linking agents, for example SMPB, which are reacted with phosphatidylethanolamine and other lipids according to literature protocols are subject to an opening of the maleimide ring during displacement of the succinimidyl group, resulting in contamination of the reacted product with the ring-opened MPB-lipid derivative. Cross-linking to proteins, antibodies, cofactors and other molecules is less than ideal using the prior art literature protocols. The method of the present invention serves to obviate this problem by providing liposomes comprising substantially pure MPB-PE, i.e., SMPB derivatized phosphatidylethanolamine exhibiting an absence of ring-opened MPB-lipid which is produced using the prior art methods. Liposomes comprising substantially pure MPB-PE may be further reacted with various proteins, for example streptavidin, among others, antibodies, cofactors and other molecules to produce conjugated liposomes of the present invention.
In accordance with a primary aspect of the present invention, i.e., the delivery of bioactive agents to a therapeutic site, the entrapment of antineoplastic agents inside liposomal bilayers has resulted in more efficacious therapy as compared to direct administration of the drug. (Forssen et.al., Cancer Res., 43, p. 546, 1983; and Gabizon et.al., Cancer Res., 42, p. 4734, 1982). A major problem with the encapsulation of antineoplastic drugs as well as other agents is that many of these drugs have been found to be rapidly released from liposomes after encapsulation. This is an undesirable effect, in view of the fact that toxicity of many of the antineoplastic agents can be significantly reduced through liposome encapsulation as compared to direct administration. See, for example, Forssen et.al. Cancer Res. 43, 546 (1983) and Rahman et.al. Cancer Res., 42, 1817 (1982). In addition, certain pharmacological agents which are favorably delivered in sustained released fashion are not accommodated by standard liposomal delivery systems; many liposomal compositions release the agent too rapidly to provide sustained release delivery.
In accordance with the present invention, a conjugated liposome made by binding a protein, antibody, cofactor or other molecule to a liposome comprised of an effective amount of substantially pure MPB-PE and related maleimide containing derivatives may be stored stably for an indefinite period, in a dehydrated state, with loading of the liposomes on an "as needed" basis.
It is an object of the present invention to provide a general method for the synthesis of substantially pure MPB-lipid and in particular, substantially pure MPB-PE and related maleimide containing derivatives and related maleimide containing derivatives and related compounds.
It is an additional object of the present invention to provide liposomes comprising substantially pure MPB-PE and related maleimide containing derivatives. Such liposomes may be further reacted with proteins, antibodies, cofactors and other molecules to produce conjugated liposomes.
It is still a further object of the present invention to provide conjugated liposomes of the present invention which have entrapped at least one bioactive agent, such as a drug.
It is still another object of the present invention to provide an efficient coupling technique in combination with stable cross-linkages to produce liposome conjugates which may more efficiently deliver encapsulated materials to cells.
It is yet an additional object of the present invention to provide stable conjugated liposomes which have more efficiently bound protein to the liposome than prior art methods and which can be stored stably for long periods of time.